A Module Swapping Approach to Engineer Allosterically Regulated Transcriptional Regulators

Allosterically regulated transcriptional regulators are key biological components for genetic response to environmental signals. These proteins bind to DNA to control gene expression, and their interactions with DNA are regulated by molecular signals. Transcriptional regulators in the LacI family are composed of two discrete and sequence modules, in which one senses molecular signals (ligand‐binding module) and the other one interacts with promoters to regulate gene expression (DNA‐binding module). Recently, we demonstrated that these LacI family modules can be interchanged robustly to generate functional regulators. We swapped ligand‐binding modules and DNA‐binding modules among LacI family members. The resulting hybrid regulators possess a new combination of allosteric response and DNA recognition properties that are originated from the ligand‐binding module and DNA‐binding module, respectively. Additionally, we showed that this module swapping approach can also be applied to regulators in the TetR family to create a hybrid protein with desirable functions. These hybrid regulators enable flexible connections between environmental signals and genetic elements, which were harnessed to implement two genetic circuit designs that were previously infeasible. These genetic circuits include a programmable ‘Passcode’ genetic device, which potentially serves as a biocontainment system to limit the proliferation of engineered cells in a highly specific environment, preventing their spread to natural environments and enhancing biosafety. As another example, we have developed a system of Multiple Toggle Switches with a Master OFF signal, which enable the equipped cells to simultaneously monitor multiple biological and environmental traits. Overall, we have established a protein design principle for creating genetic regulatory elements that facilitate cell engineering. We demonstrated that these engineered parts are sufficient for generating new cellular behaviors for potential applications in biotechnology and biomedicine. Support or Funding Information This research is supported by National Science Foundation [MCB‐1914538], the University of Texas System Rising STARs Program, and the Welch Foundation [# BP‐0037].Allosterically regulated transcriptional regulators within the same protein family have ligand‐binding modules and DNA‐binding modules that are conserved, discrete, and functional. By combining the module that responds to effector A and the module that recognizes operator B, we can create a regulator enabling the use of effector A to control activities of promoter B, which is a new input‐output connection. These engineered regulators have been used to develop several genetic networks.